Antibodies which bind specifically to activin receptor like kinase 3

ABSTRACT

A new receptor family has been identified, of activin-like kinases. Novel proteins have activin/TGF-β-type I receptor functionality, and have consequential diagnostic/therapeutic utility. They may have a serine/threonine kinase domain, a DFKSRN or DLKSKN sequence in subdomain VIB and/or a GTKRYM sequence in subdomain VIII.

RELATED APPLICATIONS

This application is a continuation application of application Ser. No.11/656,720, filed Jan. 23, 2007, now U.S. Pat. No. 7,932,351 which is adivisional of application Ser. No. 10/630,555, filed Jul. 30, 2003, nowU.S. Pat. No. 7,592,428 which is a divisional of application Ser. No.09/903,068, filed Jul. 11, 2001, now U.S. Pat. No. 6,982,319, which is adivisional application of application Ser. No. 09/679,187, filed Oct. 3,2000, now U.S. Pat. No. 6,331,621, which is a divisional application ofapplication Ser. No. 08/436,265, filed Oct. 30, 1995, now U.S. Pat. No.6,316,217, which is a 35 U.S.C. §371 of PCT/GB93/02367, filed Nov. 17,1993.

FIELD OF THE INVENTION

This invention relates to proteins having serine/threonine kinasedomains, corresponding nucleic acid molecules, and their use.

BACKGROUND OF THE INVENTION

The transforming growth factor-β (TGF-β) superfamily consists of afamily of structurally-related proteins, including three differentmammalian isoforms of TGF-β (TGF-β1, β2 and β3), activins, inhibins,mullerian-inhibiting substance and bone morphogenic proteins (BMPs) (forreviews see Roberts and Sporn, (1990) Peptide Growth Factors and TheirReceptors, Pt. 1, Sporn and Roberts, eds. (Berlin: Springer-Verlag) pp419-472; Moses et al (1990) Cell 63, 245-247). The proteins of the TGF-βsuperfamily have a wide variety of biological activities. TGF-β acts asa growth inhibitor for many cell types and appears to play a centralrole in the regulation of embryonic development, tissue regeneration,immuno-regulation, as well as in fibrosis and carcinogenesis (Robertsand Sporn (199) see above).

Activins and inhibins were originally identified as factors whichregulate secretion of follicle-stimulating hormone secretion (Vale et al(1990) Peptide Growth Factors and Their Receptors, Pt. 2, Sporn andRoberts, eds. (Berlin: Springer-Verlag) pp. 211-248). Activins were alsoshown to induce the differentiation of haematopoietic progenitor cells(Murata et al (1988) Proc. Natl. Acad. Sci. USA 85, 2434-2438; Eto et al(1987) Biochem. Biophys. Res. Commun. 142, 1095-1103) and inducemesoderm formation in Xenopus embryos (Smith et al (1990) Nature 345,729-731; van den Eijnden-Van Raaij et al (1990) Nature 345, 732-734).

BMPs or osteogenic proteins which induce the formation of bone andcartilage when implanted subcutaneously (Wozney et al (1988) Science242, 1528-1534), facilitate neuronal differentiation (Paralkar et al(1992) J. Cell Biol. 119, 1721-1728) and induce monocyte chemotaxis(Cunningham et al (1992) Proc. Natl. Acad. Sci. USA 89, 11740-11744).Müllerian-inhibiting substance induces regression of the Müllerian ductin the male reproductive system (Cate et al (1986) Cell 45, 685-698),and a glial cell line-derived neurotrophic factor enhances survival ofmidbrain dopaminergic neurons (Lin et al (1993) Science 260, 1130-1132).The action of these growth factors is mediated through binding tospecific cell surface receptors.

Within this family, TGF-β receptors have been most thoroughlycharacterized. By covalently cross-linking radio-labelled TGF-β to cellsurface molecules followed by polyacrylamide gel electrophoresis of theaffinity-labelled complexes, three distinct size classes of cell surfaceproteins (in most cases) have been identified, denoted receptor type I(53 kd), type II (75 kd), type III or betaglycan (a 300 kd proteoglycanwith a 120 kd core protein) (for a review see Massague (1992) Cell 691067-1070) and more recently endoglin (a homodimer of two 95 kdsubunits) (Cheifetz et al (1992) J. Biol. Chem. 267 19027-19030).Current evidence suggests that type I and type II receptors are directlyinvolved in receptor signal transduction (Seciarini et al (1989) Mol.Endo., 3, 261-272; Laiho et al (1991) J. Biol. Chem. 266, 9100-9112) andmay form a heteromeric complex; the type II receptor is needed for thebinding of TGF-β to the type I receptor and the type I receptor isneeded for the signal transduction induced by the type II receptor(Wrana et al (1992) Cell, 71, 1003-1004). The type III receptor andendoglin may have more indirect roles, possibly by facilitating thebinding of ligand to type II receptors (Wang et al (1991) Cell, 67797-805; Lopez-Casillas et al (1993) Cell, 73 1435-1444).

Binding analyses with activin A and BMP4 have led to the identificationof two co-existing cross-linked affinity complexes of 50-60 kDa and70-80 kDa on responsive cells (Hino et al (1989) J. Biol. Chem. 264,10309-10314; Mathews and Vale (1991), Cell 68, 775-785; Paralker et al(1991) Proc. Natl. Acad. Sci. USA 87, 8913-8917). By analogy with TGF-βreceptors they are thought to be signalling receptors and have beennamed type I and type II receptors.

Among the type II receptors for the TGF-β superfamily of proteins, thecDNA for the activin type II receptor (Act RII) was the first to becloned (Mathews and Vale (1991) Cell 65, 973-982). The predictedstructure of the receptor was shown to be a transmembrane protein withan intracellular serine/threonine kinase domain. The activin receptor isrelated to the C. elegans daf-1 gene product, but the ligand iscurrently unknown (Georgi et al (1990) Cell 61, 635-645). Thereafter,another form of the activin type II receptor (activin type IIBreceptor), of which there are different splicing variants (Mathews et al(1992), Science 225, 1702-1705; Attisano et al (1992) Cell 68, 97-108),and the TGF-β type II receptor (TβRII) (Lin et al (1992) Cell 68,775-785) were cloned, both of which have putative serine/threoninekinase domains.

SUMMARY OF THE INVENTION

The present invention involves the discovery of related novel peptides,including peptides having the activity of those defined herein as SEQ IDNos. 2, 4, 8, 10, 12, 14, 16 and 18. Their discovery is based on therealisation that receptor serine/threonine kinases form a new receptorfamily, which may include the type II receptors for other proteins inthe TGF-β superfamily. To ascertain whether there were other members ofthis family of receptors, a protocol was designed to clone ActRII/daf Irelated cDNAs. This approach made use of the polymerase chain reaction(PCR), using degenerate primers based upon the amino-acid sequencesimilarity between kinase domains of the mouse activin type II receptorand daf-I gene products.

This strategy resulted in the isolation of a new family of receptorkinases called Activin receptor like kinases (ALK's) 1-6. These cDNAsshowed an overall 33-39% sequence similarity with ActRII and TGF-β typeII receptor and 40-92% sequence similarity towards each other in thekinase domains.

Soluble receptors according to the invention comprise at leastpredominantly the extracellular domain. These can be selected from theinformation provided herein, prepared in conventional manner, and usedin any manner associated with the invention.

Antibodies to the peptides described herein may be raised inconventional manner. By selecting unique sequences of the peptides,antibodies having desired specificity can be obtained.

The antibodies may be monoclonal, prepared in known manner. Inparticular, monoclonal antibodies to the extracellular domain are ofpotential value in therapy.

Products of the invention are useful in diagnostic methods, e.g. todetermine the presence in a sample for an analyte binding therewith,such as in an antagonist assay. Conventional techniques, e.g. anenzyme-linked immunosorbent assay, may be used.

Products of the invention having a specific receptor activity can beused in therapy, e.g. to modulate conditions associated with activin orTGF-β activity. Such conditions include fibrosis, e.g. liver cirrhosisand pulmonary fibrosis, cancer, rheumatoid arthritis andglomeronephritis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the alignment of the serine/threonine (S/T) kinase domains(I-VIII) of related receptors from transmembrane proteins, includingembodiments of the present invention. The nomenclature of the subdomainsis accordingly to Hanks et al (1988). The amino acid sequences are setforth at amino acids 246-427 of SEQ ID NO: 32, 216-391 of SEQ ID NO: 31,194-368 of SEQ ID NO: 30, and 1-178 of SEQ ID NO: 33.

FIGS. 2A to 2D shows the sequences and characteristics of the respectiveprimers used in the initial PCR reactions. The nucleic acid sequencesare also given as SEQ ID Nos. 19 to 22.

FIG. 3 is a comparison of the amino-acid sequences of human activin typeII receptor (Act R-II), mouse activin type IIB receptor (Act R-IIB),human TGF-β type II receptor (TβR-II), human TGF-β type I receptor(ALK-5), human activin receptor type IA (ALK-2), and type IB (ALK-4),ALKs 1 & 3 and mouse ALK-6. See SEQ ID NOS: 30, 31, 32 10, 2, 4, 6, 8,and 18.

FIG. 4 shows, schematically, the structures for Daf-1, Act R-II, ActR-IIB, TβR-II, TβR-I/ALK-5, ALK's-1, -2 (Act RIA), -3, -4 (Act RIB) &-6.

FIG. 5 shows the sequence alignment of the cysteine-rich domains of theALKs, TβR-II, Act R-II, Act R-IIB and daf-1 receptors. See positions34-95 of SEQ ID NO: 2, 35-99 of SEQ ID NO: 4, 61-130 of SEQ ID NO: 6,34-100 of SEQ ID NO: 8, 36-106 of SEQ ID NO: 10, 30-110 of SEQ ID NO:30, 29-109 of SEQ ID NO: 31, 51-143 of SEQ ID NO: 32, and 5-101 of SEQID NO: 34.

FIG. 6 is a comparison of kinase domains of serine/threonine kinases,showing the percentage amino-acid identity of the kinase domains.

FIG. 7 shows the pairwise alignment relationship between the kinasedomains of the receptor serine/threonine kinases. The dendrogram wasgenerated using the Jotun-Hein alignment program (Hein (1990) Meth.Enzymol. 183, 626-645).

BRIEF DESCRIPTION OF THE SEQUENCE LISTINGS

Sequences 1 and 2 are the nucleotide and deduced amino-acid sequences ofcDNA for hALK-1 (clone HP57).

Sequences 3 and 4 are the nucleotide and deduced amino-acid sequences ofcDNA for hALK-2 (clone HP53).

Sequences 5 and 6 are the nucleotide and deduced amino-acid sequences ofcDNA for hALK-3 (clone ONF5).

Sequences 7 and 8 are the nucleotide and deduced amino-acid sequences ofcDNA for hALK-4 (clone 11H8), complemented with PCR product encodingextracellular domain.

Sequences 9 and 10 are the nucleotide and deduced amino-acid sequencesof cDNA for hALK-5 (clone EMBLA).

Sequences 11 and 12 are the nucleotide and deduced amino-acid sequencesof cDNA for mALK-1 (clone AM6).

Sequences 13 and 14 are the nucleotide and deduced amino-acid sequencesof cDNA for mALK-3 (clones ME-7 and ME-D).

Sequences 15 and 16 are the nucleotide and deduced amino-acid sequencesof cDNA for mALK-4 (clone 8a1).

Sequences 17 and 18 are the nucleotide and deduced amino-acid sequencesof cDNA for mALK-6 (clone ME-6).

Sequence 19 (B1-S) is a sense primer, extracellular domain,cysteine-rich region, BamHI site at 5′ end, 28-mer, 64-fold degeneracy.

Sequence 20 (B3-S) is a sense primer, kinase domain II, BamHI site at 5′end, 25-mer, 162-fold degeneracy.

Sequence 21 (B7-S) is a sense primer, kinase domain VIB, S/T kinasespecific residues, BamHI site at 5′ end, 24-mer, 288-fold degeneracy.

Sequence 22 (E8-AS) is an anti-sense primer, kinase domain, S/Tkinase-specific residues EcoRI site at 5′ end, 20-mer, 18-folddegeneracy.

Sequence 23 is an oligonucleotide probe.

Sequence 24 is a 5′ primer.

Sequence 25 is a 3′ primer.

Sequence 26 is a consensus sequence in Subdomain I.

Sequences 27 and 28 are novel sequence motifs in Subdomain VIB.

Sequence 29 is a novel sequence motif in Subdomain VIII.

DESCRIPTION OF THE INVENTION

As described in more detail below, nucleic acid sequences have beenisolated, coding for a new sub-family of serine/threonine receptorkinases. The term nucleic acid molecules as used herein refers to anysequence which codes for the murine, human or mammalian form, amino-acidsequences of which are presented herein. It is understood that the wellknown phenomenon of codon degeneracy provides for a great deal ofsequence variation and all such varieties are included within the scopeof this invention.

The nucleic acid sequences described herein may be used to clone therespective genomic DNA sequences in order to study the genes' structureand regulation. The murine and human cDNA or genomic sequences can alsobe used to isolate the homologous genes from other mammalian species.The mammalian DNA sequences can be used to study the receptors'functions in various in vitro and in vivo model systems.

As exemplified below for ALK-5 cDNA, it is also recognised that, giventhe sequence information provided herein, the artisan could easilycombine the molecules with a pertinent promoter in a vector, so as toproduce a cloning vehicle for expression of the molecule. The promoterand coding molecule must be operably linked via any of thewell-recognized and easily-practised methodologies for so doing. Theresulting vectors, as well as the isolated nucleic acid moleculesthemselves, may be used to transform prokaryotic cells (e.g. E. coli),or transfect eukaryotes such as yeast (S. cerevisiae), PAE, COS or CHOcell lines. Other appropriate expression systems will also be apparentto the skilled artisan.

Several methods may be used to isolate the ligands for the ALKs. Asshown for ALK-5 cDNA, cDNA clones encoding the active open readingframes can be subcloned into expression vectors and transfected intoeukaryotic cells, for example COS cells. The transfected cells which canexpress the receptor can be subjected to binding assays forradioactively-labelled members of the TGF-β superfamily (TGF-β,activins, inhibins, bone morphogenic proteins and mullerian-inhibitingsubstances), as it may be expected that the receptors will bind membersof the TGF-β superfamily. Various biochemical or cell-based assays canbe designed to identify the ligands, in tissue extracts or conditionedmedia, for receptors in which a ligand is not known. Antibodies raisedto the receptors may also be used to identify the ligands, using theimmunoprecipitation of the cross-linked complexes. Alternatively,purified receptor could be used to isolate the ligands using anaffinity-based approach. The determination of the expression patterns ofthe receptors may also aid in the isolation of the ligand. These studiesmay be carried out using ALK DNA or RNA sequences as probes to performin situ hybridisation studies.

The use of various model systems or structural studies should enable therational development of specific agonists and antagonists useful inregulating receptor function. It may be envisaged that these can bepeptides, mutated ligands, antibodies or other molecules able tointeract with the receptors.

The foregoing provides examples of the invention Applicants intend toclaim which includes, inter alia, isolated nucleic acid molecules codingfor activin receptor-like kinases (ALKs), as defined herein. Theseinclude such sequences isolated from mammalian species such as mouse,human, rat, rabbit and monkey.

The following description relates to specific embodiments. It will beunderstood that the specification and examples are illustrative but notlimitative of the present invention and that other embodiments withinthe spirit and scope of the invention will suggest themselves to thoseskilled in the art.

Preparation of mRNA and Construction of a cDNA Library

For construction of a cDNA library, poly (A)⁺ RNA was isolated from ahuman erythroleukemia cell line (HEL 92.1.7) obtained from the AmericanType Culture Collection (ATCC TIB 180). These cells were chosen as theyhave been shown to respond to both activin and TGF-β. Moreover leukaemiccells have proved to be rich sources for the cloning of novel receptortyrosine kinases (Partanen et al (1990) Proc. Natl. Acad. Sci. USA 87,8913-8917 and (1992) Mol. Cell. Biol. 12, 1698-1707). (Total) RNA wasprepared by the guanidinium isothiocyanate method (Chirgwin et al (1979)Biochemistry 18, 5294-5299). mRNA was selected using the poly-A or polyAT tract mRNA isolation kit (Promega, Madison, Wis., U.S.A.) asdescribed by the manufacturers, or purified through an oligo(dT)-cellulose column as described by Aviv and Leder (1972) Proc. Natl.Acad. Sci. USA 69, 1408-1412. The isolated mRNA was used for thesynthesis of random primed (Amersham) cDNA, that was used to make aλgt10 library with 1×10⁵ independent cDNA clones using the RiboclonecDNA synthesis system (Promega) and λgt10 in vitro packaging kit(Amersham) according to the manufacturers' procedures. An amplifiedoligo (dT) primed human placenta λZAPII cDNA library of 5×10⁵independent clones was used. Poly (A)⁺ RNA isolated from AG1518 humanforeskin fibroblasts was used to prepare a primary random primed λZAPIIcDNA library of 1.5×10⁶ independent clones using the RiboClone cDNAsynthesis system and Gigapack Gold II packaging extract (Stratagene). Inaddition, a primary oligo (dT) primed human foreskin fibroblast λgt10cDNA library (Claesson-Welsh et al (1989) Proc. Natl. Acad. Sci. USA. 864917-4912) was prepared. An amplified oligo (dT) primed HEL cell λgt11cDNA library of 1.5×10⁶ independent clones (Poncz et al (1987) Blood 69219-223) was used. A twelve-day mouse embryo λEXIox cDNA library wasobtained from Novagen (Madison, Wis., U.S.A.); a mouse placenta λZAPIIcDNA library was also used.

Generation of cDNA Probes by PCR

For the generation of cDNA probes by PCR (Lee et al (1988) Science 239,1288-1291) degenerate PCR primers were constructed based upon theamino-acid sequence similarity between the mouse activin type IIreceptor (Mathews and Vale (1991) Cell 65, 973-982) and daf-1 (George etal (1990) Cell 61, 635-645) in the kinase domains II and VIII. FIG. 1shows the aligned serine/threonine kinase domains (1-VIII), of fourrelated receptors of the TGF-β superfamily, i.e. hTβR-II, mActR-IIB,mActR-II and the daf-1 gene product, using the nomenclature of thesubdomains according to Hanks et al (1988) Science 241, 45-52.

Several considerations were applied in the design of the PCR primers.The sequences were taken from regions of homology between the activintype II receptor and the daf-1 gene product, with particular emphasis onresidues that confer serine/threonine specificity (see Table 2) and onresidues that are shared by transmembrane kinase proteins and not bycytoplasmic kinases. The primers were designed so that each primer of aPCR set had an approximately similar GC composition, and so that selfcomplementarity and complementarity between the 3′ ends of the primersets were avoided. Degeneracy of the primers was kept as low aspossible, in particular avoiding serine, leucine and arginine residues(6 possible codons), and human codon preference was applied. Degeneracywas particularly avoided at the 3′ end as, unlike the 5′ end, wheremismatches are tolerated, mismatches at the 3′ end dramatically reducethe efficiency of PCR.

In order to facilitate directional subcloning, restriction enzyme siteswere included at the 5′ end of the primers, with a GC clamp, whichpermits efficient restriction enzyme digestion. The primers utilised areshown in FIG. 2. Oligonucleotides were synthesized using Gene assemblerplus (Pharmacia—LKB) according to the manufacturers instructions.

The mRNA prepared from HEL cells as described above wasreverse-transcribed into cDNA in the presence of 50 mM Tris-HCl, pH 8.3,8 mM MgCl₂, 30 mM KCl, 10 mM dithiothreitol, 2 mM nucleotidetriphosphates, excess oligo (dT) primers and 34 units of AMV reversetranscriptase at 42° C. for 2 hours in 40 μl of reaction volume.Amplification by PCR was carried out with a 7.5% aliquot (3 μl) of thereverse-transcribed mRNA, in the presence of 10 mM Tris-HCl, pH 8.3, 50mM KCl, 1.5 M MgCl₂, 0.01% gelatin, 0.2 mM nucleotide triphosphates, 1μM of both sense and antisense primers and 2.5 units of Taq polymerase(Perkin Elmer Cetus) in 100 μl reaction volume. Amplifications wereperformed on a thermal cycler (Perkin Elmer Cetus) using the followingprogram: first 5 thermal cycles with denaturation for 1 minute at 94°C., annealing for 1 minute at 50° C., a 2 minute ramp to 55° C. andelongation for 1 minute at 72° C., followed by 20 cycles of 1 minute at94° C., 30 seconds at 55° C. and 1 minute at 72° C. A second round ofPCR was performed with 3 μl of the first reaction as a template. Thisinvolved 25 thermal cycles, each composed of 94° C. (1 min), 55° C. (0.5min), 72° C. (1 min).

General procedures such as purification of nucleic acids, restrictionenzyme digestion, gel electrophoresis, transfer of nucleic acid to solidsupports and subcloning were performed essentially according toestablished procedures as described by Sambrook et al, (1989), Molecularcloning: A Laboratory Manual, 2^(nd) Ed. Cold Spring Harbor Laboratory(Cold Spring Harbor, N.Y., USA).

Samples of the PCR products were digested with BamHI and EcoRI andsubsequently fractionated by low melting point agarose gelelectrophoresis. Bands corresponding to the approximate expected sizes,(see Table 1: ≈460 bp for primer pair B3-S and E8-AS and ≈140 bp forprimer pair B7-S and E8-AS) were excised from the gel and the DNA waspurified. Subsequently, these fragments were ligated into pUC19(Yanisch-Perron et al (1985) Gene 33, 103-119), which had beenpreviously linearised with BamHI and EcoRI and transformed into E. colistrain DH5α using standard protocols (Sambrook et al, supra). Individualclones were sequenced using standard double-stranded sequencingtechniques and the dideoxynucleotide chain termination method asdescribed by Sanger et al (1977) Proc. Natl. Acad. Sci. USA 74,5463-5467, and T7 DNA polymerase.

Employing Reverse Transcriptase PCR on HEL mRNA with the primer pairB3-S and E8-AS, three PCR products were obtained, termed 11.1, 11.2 and11.3, that corresponded to novel genes. Using the primer pair B7-S andE8-AS, an additional novel PCR product was obtained termed 5.2.

TABLE 1 SEQUENCE SITE OF DNA SEQUENCE IDENTITY FRAGMENT IN IDENTITY WITHBETWEEN NAME OF INSERT mActRII/hTBRII SEQUENCE mActRII PCR SITE CLONESmActRII/hTBRII and TBR- PRODUCT PRIMERS (bp) (bp) (%) 11 (%) 11.1B3-S/E8- 460 460 46/40 42 AS 11.2 B3-S/E8- 460 460 49/44 47 AS 11.3B3-S/E8- 460 460 44/36 48 AS 11.29 B3-S/E8- 460 460 ND/100 ND AS 9.2B1-S/E8- 800 795 100/ND ND AS 5.2 B7-S/E8- 140 143 40/38 60 ASIsolation of cDNA Clones

The PCR products obtained were used to screen various cDNA librariesdescribed supra. Labelling of the inserts of PCR products was performedusing random priming method (Feinberg and Vogelstein (1983) Anal.Biochem, 132 6-13) using the Megaprime DNA labelling system (Amersham).The oligonucleotide derived from the sequence of the PCR product 5.2 waslabelled by phosphorylation with T4 polynucleotide kinase followingstandard protocols (Sambrook et al, supra). Hybridization andpurification of positive bacteriophages were performed using standardmolecular biological techniques.

The double-stranded DNA clones were all sequenced using thedideoxynucleotide chain-termination method as described by Sanger et al,supra, using T7 DNA polymerase (Pharmacia—LKB) or Sequenase (U.S.Biochemical Corporation, Cleveland, Ohio, U.S.A.). Compressions ofnucleotides were resolved using 7-deaza-GTP (U.S. Biochemical Corp.) DNAsequences were analyzed using the DNA STAR computer program (DNA STARLtd. U.K.). Analyses of the sequences obtained revealed the existence ofsix distinct putative receptor serine/threonine kinases which have beennamed ALK 1-6.

To clone cDNA for ALK-1 the oligo (dT) primed human placenta cDNAlibrary was screened with a radiolabelled insert derived from the PCRproduct 11.3; based upon their restriction enzyme digestion patterns,three different types of clones with approximate insert sizes of 1.7 kb,2 kb & 3.5 kb were identified. The 2 kb clone, named HP57, was chosen asrepresentative of this class and subjected to complete sequencing.Sequence analysis of ALK-1 revealed a sequence of 1984 nucleotidesincluding a poly-A tail (SEQ ID No. 1). The longest open reading frameencodes a protein of 503 amino-acids, with high sequence similarity toreceptor serine/threonine kinases (see below). The first methioninecodon, the putative translation start site, is at nucleotide 283-285 andis preceded by an in-frame stop codon. This first ATG is in a morefavourable context for translation initiation (Kozak (1987) Nucl. AcidsRes., 15, 8125-8148) than the second and third in-frame ATG atnucleotides 316-318 and 325-327. The putative initiation codon ispreceded by a 5′ untranslated sequence of 282 nucleotides that isGC-rich (80% GC), which is not uncommon for growth factor receptors(Kozak (1991) J. Cell Biol., 115, 887-903). The 3′ untranslated sequencecomprises 193 nucleotides and ends with a poly-A tail. No bona fidepoly-A addition signal is found, but there is a sequence (AATACA), 17-22nucleotides upstream of the poly-A tail, which may serve as a poly-Aaddition signal.

ALK-2 cDNA was cloned by screening an amplified oligo (dT) primed humanplacenta cDNA library with a radiolabelled insert derived from the PCRproduct 11.2. Two clones, termed HP53 and HP64, with insert sizes of 2.7kb and 2.4 kb respectively, were identified and their sequences weredetermined. No sequence difference in the overlapping clones was found,suggesting they are both derived from transcripts of the same gene.

Sequence analysis of cDNA clone HP53 (SEQ ID No. 3) revealed a sequenceof 2719 nucleotides with a poly-A tail. The longest open reading frameencodes a protein of 509 amino-acids. The first ATG at nucleotides104-106 agrees favourably with Kozak's consensus sequence with an A atposition 3. This ATG is preceded in-frame by a stop codon. There arefour ATG codons in close proximity further downstream, which agree withthe Kozak's consensus sequence (Kozak, supra), but according to Kozak'sscanning model the first ATG is predicted to be the translation startsite. The 5′ untranslated sequence is 103 nucleotides. The 3′untranslated sequence of 1089 nucleotides contains a polyadenylationsignal located 9-14 nucleotides upstream from the poly-A tail. The cDNAclone HP64 lacks 498 nucleotides from the 5′ end compared to HP53, butthe sequence extended at the 3′ end with 190 nucleotides and poly-A tailis absent. This suggests that different polyadenylation sites occur forALK-2. In Northern blots, however, only one transcript was detected (seebelow).

The cDNA for human ALK-3 was cloned by initially screening an oligo (dT)primed human foreskin fibroblast cDNA library with an oligonucleotide(SEQ ID No. 23) derived from the PCR product 5.2. One positive cDNAclone with an insert size of 3 kb, termed 0N11, was identified. However,upon partial sequencing, it appeared that this clone was incomplete; itencodes only part of the kinase domain and lacks the extracelluardomain. The most 5′ sequence of ON11, a 540 nucleotide XbaI restrictionfragment encoding a truncated kinase domain, was subsequently used toprobe a random primed fibroblast cDNA library from which one cDNA clonewith an insert size of 3 kb, termed 0NF5, was isolated (SEQ ID No. 5).Sequence analysis of ONF5 revealed a sequence of 2932 nucleotideswithout a poly-A tail, suggesting that this clone was derived byinternal priming. The longest open reading frame codes for a protein of532 amino-acids. The first ATG codon which is compatible with Kozak'sconsensus sequence (Kozak, supra), is at 310-312 nucleotides and ispreceded by an in-frame stop codon. The 5′ and 3′ untranslated sequencesare 309 and 1027 nucleotides long, respectively.

ALK-4 cDNA was identified by screening a human oligo (dT) primed humanerythroleukemia cDNA library with the radiolabelled insert of the PCRproduct 11.1 as a probe. One cDNA clone, termed 11H8, was identifiedwith an insert size of 2 kb (SEQ ID No. 7). An open reading frame wasfound encoding a protein sequence of 383 amino-acids encoding atruncated extracellular domain with high similarity to receptorserine/threonine kinases. The 3′ untranslated sequence is 818nucleotides and does not contain a poly-A tail, suggesting that the cDNAwas internally primed. cDNA encoding the complete extracellular domain(nucleotides 1-366) was obtained from HEL cells by RT-PCR with 5′ primer(SEQ ID No. 24) derived in part from sequence at translation start siteof SKR-2 (a cDNA sequence deposited in GenBank data base, accesionnumber L10125, that is identical in part to ALK-4) and 3′ primer (SEQ IDNo. 25) derived from 11H8 cDNA clone.

ALK-5 was identified by screening the random primed HEL cell λgt 10 cDNAlibrary with the PCR product 11.1 as a probe. This yielded one positiveclone termed EMBLA (insert size of 5.3 kb with 2 internal EcoRI sites).Nucleotide sequencing revealed an open reading frame of 1509 bp, codingfor 503 amino-acids. The open reading frame was flanked by a 5′untranslated sequence of 76 bp, and a 3′ untranslated sequence of 3.7 kbwhich was not completely sequenced. The nucleotide and deducedamino-acid sequences of ALK-5 are shown in SEQ ID Nos. 9 and 10. In the5′ part of the open reading frame, only one ATG codon was found; thiscodon fulfils the rules of translation initiation (Kozak, supra). Anin-frame stop codon was found at nucleotides (−54)-(−52) in the 5′untranslated region. The predicted ATG start codon is followed by astretch of hydrophobic amino-acid residues which has characteristics ofa cleavable signal sequence. Therefore, the first ATG codon is likely tobe used as a translation initiation site. A preferred cleavage site forthe signal peptidase, according to von Heijne (1986) Nucl. Acid. Res.14, 4683-4690, is located between amino-acid residues 24 and 25. Thecalculated molecular mass of the primary translated product of the ALK-5without signal sequence is 53,646 Da.

Screening of the mouse embryo λEX Iox cDNA library using PCR, product11.1 as a probe yielded 20 positive clones. DNAs from the positiveclones obtained from this library were digested with EcoRI and HindIII,electrophoretically separated on a 1.3% agarose gel and transferred tonitrocellulose filters according to established procedures as describedby Sambrook et al, supra. The filters were then hybridized with specificprobes for human ALK-1 (nucleotide 288-670), ALK-2 (nucleotide 1-581),ALK-3 (nucleotide 79-824) or ALK-4 nucleotide 1178-1967). Such analysesrevealed that a clone termed ME-7 hybridised with the human ALK-3 probe.However, nucleotide sequencing revealed that this clone was incomplete,and lacked the 5′ part of the translated region. Screening the same cDNAlibrary with a probe corresponding to the extracelluar domain of humanALK-3 (nucleotides 79-824) revealed the clone ME-D. This clone wasisolated and the sequence was analyzed. Although this clone wasincomplete in the 3′ end of the translated region, ME-7 and ME-Doverlapped and together covered the complete sequence of mouse ALK-3.The predicted amino-acid sequence of mouse ALK-3 is very similar to thehuman sequence; only 8 amino-acid residues differ (98% identity; see SEQID No. 14) and the calculated molecular mass of the primary translatedproduct without the putative signal sequence is 57,447 Da.

Of the clones obtained from the initial library screening with PCRproduct 11.1, four clones hybridized to the probe corresponding to theconserved kinase domain of ALK-4 but not to probes from more divergentparts of ALK-1 to -4. Analysis of these clones revealed that they havean identical sequence which differs from those of ALK-1 to -5 and wastermed ALK-6. The longest clone ME6 with a 2.0 kb insert was completelysequenced yielding a 1952 bp fragment consisting of an open readingframe of 1506 bp (502 amino-acids), flanked by a 5′ untranslatedsequence of 186 bp, and a 3′ untranslated sequence of 160 bp. Thenucleotide and predicted amino-acid sequences of mouse ALK-6 are shownin SEQ ID Nos. 17 and 18. No polyadenylation signal was found in the 3′untranslated region of ME6, indicating that the cDNA was internallyprimed in the 3′ end. Only one ATG codon was found in the 5′ part of theopen reading frame, which fulfils the rules for translation initiation(Kozak, supra), and was preceded by an in-frame stop codon atnucleotides 163-165. However, a typical hydrophobic leader sequence wasnot observed at the N terminus of the translated region. Since there isno ATG codon and putative hydrophobic leader sequence, this ATG codon islikely to be used as a translation initiation site. The calculatedmolecular mass of the primary translated product with the putativesignal sequence is 55,576 Da.

Mouse ALK-1 (clone AM6 with 1.9 kb insert) was obtained from the mouseplacenta λZAPII cDNA library using human ALK-1 cDNA as a probe (see SEQID No. 11). Mouse ALK-4 (clone 8a1 with 2.3 kb insert) was also obtainedfrom this library using human ALK-4 cDNA library as a probe (SEQ ID No.15).

To summarise, clones HP22, HP57, ONF1, ONF3, ONF4 and HP29 encode thesame gene, ALK-1. Clone AM6 encodes mouse ALK-1. HP53, HP64 and HP84encode the same gene, ALK-2. ONF5, ONF2 and ON11 encode the same geneALK-3. ME-7 and ME-D encode the mouse counterpart of human ALK-3. 11H8encodes a different gene ALK-4, whilst 8aI encodes the mouse equivalent.EMBLA encodes ALK-5, and ME-6 encodes ALK-6.

The sequence alignment between the 6 ALK genes and TβR-II, mActR-II andActR-IIB is shown in FIG. 3. These molecules have a similar domainstructure; an N-terminal predicted hydrophobic signal sequence (vonHeijne (1986) Nucl. Acids Res. 14: 4683-4690) is followed by arelatively small extracellular cysteine-rich ligand binding domain, asingle hydrophobic transmembrane region (Kyte & Doolittle (1982) J. Mol.Biol. 157, 105-132) and a C-terminal intracellular portion, whichconsists almost entirely of a kinase domain (FIGS. 3 and 4).

The extracelluar domains of these receptors have cysteine-rich regions,but they show little sequence similarity; for example, less than 20%sequence identity is found between Daf-1, ActR-II, TBR-II and ALK-5. TheALKs appear to form a subfamily as they show higher sequencesimilarities (15-47% identity) in their extracellular domains. Theextracellular domains of ALK-5 and ALK-4 have about 29% sequenceidentity. In addition, ALK-3 and ALK-6 share a high degree of sequencesimilarity in their extracellular domains (46% identity).

The positions of many of the cysteine residues in all receptors can bealigned, suggesting that the extracellular domains may adopt a similarstructural configuration. See FIG. 5 for ALKs-1, -2, -3 & -5. Each ofthe ALKs (except ALK-6) has a potential N-linked glycosylation site, theposition of which is conserved between ALK-1 and ALK-2, and betweenALK-3, ALK-4 and ALK-5 (see FIG. 4).

The sequence similarities in the kinase domains between daf-1, ActR-II,TβR-II and ALK-5 are approximately 40%, whereas the sequence similaritybetween the ALKs 1 to 6 is higher (between 59% and 90%; see FIG. 6).Pairwise comparison using the Jutun-Hein sequence alignment program(Hein (1990) Meth, Enzymol., 183, 626-645), between all family members,identifies the ALKs as a separate subclass among serine/threoninekinases (FIG. 7).

The catalytic domains of kinases can be divided into 12 subdomains withstretches of conserved amino-acid residues. The key motifs are found inserine/threonine kinase receptors suggesting that they are functionalkinases. The consensus sequence for the binding of ATP(Gly-X-Gly-X-X-Gly in subdomain I followed by a Lys residue furtherdownstream in subdomain II) is found in all the ALKs.

The kinase domains of daf-1, ActR-II, and ALKs show approximately equalsequence similarity with tyrosine and serine/threonine protein kinases.However analysis of the amino-acid sequences in subdomains VI and VIII,which are the most useful to distinguish a specificity forphosphorylation of tyrosine residues versus serine/threonine residues(Hanks et al (1988) Science 241 42-52) indicates that these kinases areserine/threonine kinases; refer to Table 2.

TABLE 2 SUBDOMAINS (SEQ ID NOS: ) KINASE VIB VIII Serine/threonineDLKPEN G (T/S) XX  kinase consensus 35 (Y/F) X 37-40 Tyrosine DLAAFNXP (I/V) kinase consensus 36 (K/R) W (T/M) 41-48 Act R-II DIKSKN GTRRYMAmino acids  Amino acids  322-327 of 361-366 of SEQ ID NO: 30SEQ ID NO: 30 Act R-III DFKSKN GTRRYM Amino acids  Amino acids 345-350 of 361-366 of SEQ ID NO: 31 SEQ ID NO: 31 TyR-II DLKSSN GTARYMAmino acids  Amino acids  379-384 of 420-425 of SEQ ID NO: 32 SEQ ID NO: 32 ALK-I DFKSRN GTKRYM Amino acids  29 330-335 ofSEQ ID NO: 3 ALK-2, -3, -4, DLKSKN GTKRYM -5, & -6 28 29

The sequence motifs DLKSKN (Subdomain VIB) and GTKRYM (Subdomain VIII),that are found in most of the serine/threonine kinase receptors, agreewell with the consensus sequences for all protein serine/threoninekinase receptors in these regions. In addition, these receptors, exceptfor ALK-1, do not have a tyrosine residue surrounded by acidic residuesbetween subdomains VII and VIII, which is common for tyrosine kinases. Aunique characteristic of the members of the ALK serine/threonine kinasereceptor family is the presence of two short inserts in the kinasedomain between subdomains VIA and VIB and between subdomains X and XI.In the intracellular domain, these regions, together with thejuxtamembrane part and C-terminal tail, are the most divergent betweenfamily members (see FIGS. 3 and 4). Based on the sequence similaritywith the type II receptors for TGF-β and activin, the C termini of thekinase domains of ALKs-1 to -6 are set at Ser-495, Ser-501, Ser-527,Gln-500, Gln-498 and Ser-497, respectively.

mRNA Expression

The distribution of ALK-1, -2, -3, -4 was determined by Northern blotanalysis. A Northern blot filter with mRNAs from different human tissueswas obtained from Clontech (Palo Alto, Calif.). The filters werehybridized with ³²P-labelled probes at 42° C. overnight in 50%formaldehyde, 5× standard saline citrate (SSC; 1×SSC is 50 mM sodiumcitrate, pH 7.0, 150 mM NaCl), 0.1% SDS, 50 mM sodium phosphate,5×Denhardt's solution and 0.1 mg/ml salmon sperm DNA. In order tominimize cross-hybridization, probes were used that did not encode partof the kinase domains, but corresponded to the highly diverged sequencesof either 5′ untranslated and ligand-binding regions (probes for ALK-1,-2 and -3) or 3′ untranslated sequences (probe for ALK-4). The probeswere labelled by random priming using the Multiprime (or Mega-prime) DNAlabelling system and [α-³²P] dCTP (Feinberg & Vogelstein (1983) Anal.Biochem. 132: 6-13). Unincorporated label was removed by Sephadex G-25chromatography. Filters were washed at 65° C., twice for 30 minutes in2.5×SSC, 0.1% SDS and twice for 30 minutes in 0.3×SSC, 0.1% SDS beforebeing exposed to X-ray film. Stripping of blots was performed byincubation at 90-100° C. in water for 20 minutes.

The ALK-5 mRNA size and distribution were determined by Northern blotanalysis as above. An EcoRl fragment of 980 bp of the full length ALK-5cDNA clone, corresponding to the C-terminal part of the kinase domainand 3′ untranslated region (nucleotides 1259-2232 in SEQ ID No. 9) wasused as a probe. The filter was washed twice in 0.5×SSC, 0.1% SDS at 55°C. for 15 minutes.

Using the probe for ALK-1, two transcripts of 2.2 and 4.9 kb weredetected. The ALK-1 expression level varied strongly between differenttissues, high in placenta and lung, moderate in heart, muscle andkidney, and low (to not detectable) in brain, liver and pancreas. Therelative ratios between the two transcripts were similar in mosttissues; in kidney, however, there was relatively more of the 4.9 kbtranscript. By reprobing the blot with a probe for ALK-2, one transcriptof 4.0 kb was detected with a ubiquitous expression pattern. Expressionwas detected in every tissue investigated and was highest in placentaand skeletal muscle. Subsequently the blot was reprobed for ALK-3. Onemajor transcript of 4.4 kb and a minor transcript of 7.9 kb weredetected. Expression was high in skeletal muscle, in which also anadditional minor transcript of 10 kb was observed. Moderate levels ofALK-3 mRNA were detected in heart, placenta, kidney and pancreas, andlow (to not detectable) expression was found in brain, lung and liver.The relative ratios between the different transcripts were similar inthe tested tissues, the 4.4 kb transcript being the predominant one,with the exception for brain where both transcripts were expressed at asimilar level. Probing the blot with ALK-4 indicated the presence of atranscript with the estimated size of 5.2 kb and revealed an ubiquitousexpression pattern. The results of Northern blot analysis using theprobe for ALK-5 showed that a 5.5 kb transcript is expressed in allhuman tissues tested, being most abundant in placenta and least abundantin brain and heart.

The distribution of mRNA for mouse ALK-3 and -6 in various mouse tissueswas also determined by Northern blot analysis. A multiple mouse tissueblot was obtained from Clontech, Palo Alto, Calif., U.S.A. The filterwas hybridized as described above with probes for mouse ALK-3 and ALK-6.The EcoRI-PstI restriction fragment, corresponding to nucleotides79-1100 of ALK-3, and the SacI-HpaI fragment, corresponding tonucleotides 57-720 of ALK-6, were used as probes. The filter was washedat 65° C. twice for 30 minutes in 2.5×SSC, 0.1% SDS and twice for 30minutes with 0.3×SSC, 0.1% SDS and then subjected to autoradiography.

Using the probe for mouse ALK-3, a 1.1 kb transcript was found only inspleen. By reprobing the blot with the ALK-6 specific probe, atranscript of 7.2 kb was found in brain and a weak signal was also seenin lung. No other signal was seen in the other tissues tested, i.e.heart, liver, skeletal muscle, kidney and testis.

All detected transcript sizes were different, and thus no cross-reactionbetween mRNAs for the different ALKs was observed when the specificprobes were used. This suggests that the multiple transcripts of ALK-1and ALK-3 are coded from the same gene. The mechanism for generation ofthe different transcripts is unknown at present; they may be formed byalternative mRNA splicing, differential polyadenylation, use ofdifferent promotors, or by a combination of these events. Differences inmRNA splicing in the regions coding for the extracellular domains maylead to the synthesis of receptors with different affinities forligands, as was shown for mActR-IIB (Attisano et al (1992) Cell 68,97-108) or to the production of soluble binding protein.

The above experiments describe the isolation of nucleic acid sequencescoding for new family of human receptor kinases. The cDNA for ALK-5 wasthen used to determine the encoded protein size and binding properties.

Properties of the ALKs cDNA Encoded Proteins

To study the properties of the proteins encoded by the different ALKcDNAs, the cDNA for each ALK was subcloned into a eukaryotic expressionvector and transfected into various cell types and then subjected toimmunoprecipitation using a rabbit antiserum raised against a syntheticpeptide corresponding to part of the intracellular juxtamembrane region.This region is divergent in sequence between the variousserine/threonine kinase receptors. The following amino-acid residueswere used:

ALK-1 145-166 ALK-2 151-172 ALK-3 181-202 ALK-4 153-171 ALK-5 158-179ALK-6 151-168

The rabbit antiseru against ALK-5 was designated VPN.

The peptides were synthesized with an Applied Biosystems 430A PeptideSynthesizer using t-butoxycarbonyl chemistry and purified byreversed-phase high performance liquid chromatography. The peptides werecoupled to keyhole limpet haemocyanin (Calbiochem-Behring) usingglutaraldehyde, as described by Guillick et al (1985) EMBO J. 4,2869-2877. The coupled peptides were mixed with Freunds adjuvant andused to immunize rabbits.

Transient Transfection of the ALK-5 cDNA

COS-1 cells (American Type Culture Collection) and the R mutant of MvlLucells (for references, see below) were cultured in Dulbecco's modifiedEagle's medium containing 10% fetal bovine serum (FBS) and 100 units/mlpenicillin and 50 μg 1 ml streptomycin in 5% C0₂ atmosphere at 37° C.The ALK-5 cDNA (nucleotides (−76)-2232), which includes the completecoding region, was cloned in the pSV7d vector (Truett et al, (1985) DNA4, 333-349), and used for transfection. Transfection into COS-1 cellswas performed by the calcium phosphate precipitation method (Wigler etal (1979) Cell 16, 777-785). Briefly, cells were seeded into 6-well cellculture plates at a density of 5×10⁵ cells/well, and transfected thefollowing day with 10 μg of recombinant plasmid. After overnightincubation, cells were washed three times with a buffer containing 25 mMTris-HCl, pH 7.4, 138 mM NaCl, 5 mM KCl, 0.7 mM CaCl₂, 0.5 mM MgCl₂ and0.6 mM Na₂HPO₄, and then incubated with Dulbecco's modified Eagle'smedium containing FBS and antibiotics. Two days after transfection, thecells were metabolically labelled by incubating the cells for 6 hours inmethionine and cysteine-free MCDB 104 medium with 150 μCi/ml of[³⁵S]-methionine and [³⁵S]-cysteine (in vivo labelling mix; Amersham).After labelling, the cells were washed with 150 mM NaCl, 25 mM Tris-HCl,pH 7.4, and then solubilized with a buffer containing 20mM Tris-HCl, pH7.4, 150 mM NaCl, 10 mM EDTA, 1% Triton X-100, 1% deoxycholate, 1.5%Trasylol (Bayer) and 1 mM phenylmethylsulfonylfluoride (PMSF; Sigma).After 15 minutes on ice, the cell lysates were pelleted bycentrifugation, and the supernatants were then incubated with 7 μl ofpreimmune serum for 1.5 hours at 4° C. Samples were then given 50 μl ofprotein A-Sepharose (Pharmacia-LKB) slurry (50% packed beads in 150 mMNaCl, 20 mM Tris-HCl, pH 7.4, 0.2% Triton X100) and incubated for 45minutes at 4° C. The beads were spun down by centrifugation, and thesupernatants (1 ml) were then incubated with either 7 μl of preimmuneserum or the VPN antiserum for 1.5 hours at 4° C. For blocking, 10 μg ofpeptide was added together with the antiserum. Immune complexes werethen given 50 μl of protein A-Sepharose (Pharmacia—LKB) slurry (50%packed beads in 150 mM NaCl, 20mM Tris-HCl, pH 7.4, 0.2% Triton X-100)and incubated for 45 minutes at 4° C. The beads were spun down andwashed four times with a washing buffer (20 mM Tris-HCl, pH 7.4, 500 mMNaCl, 1% Triton X-100, 1% deoxycholate and 0.2% SDS), followed by onewash in distilled water. The immune complexes were eluted by boiling for5 minutes in the SDS-sample buffer (100 mM Tris-HCl, pH 8.8, 0.01%bromophenol blue, 36% glycerol, 4% SDS) in the presence of 10 mM DTT,and analyzed by SDS-gel electrophoresis using 7-15% polyacrylamide gels(Blobel and Dobberstein, (1975) J. Cell Biol. 67, 835-851). Gels werefixed, incubated with Amplify (Amersham) for 20 minutes, and subjectedto fluorography. A component of 53 Da was seen. This component was notseen when preimmune serum was used, or when 10 μg blocking peptide wasadded together with the antiserum. Moreover, it was not detectable insamples derived from untransfected COS-1 cells using either preimmuneserum or the antiserum.

Digestion with Endoglycosidase F

Samples immunoprecipitated with the VPN antisera obtained as describedabove were incubated with 0.5 U of endoglycosidase F (BoehringerMannheim Biochemica) in a buffer containing 100 mM sodium phosphate, pH6.1, 50 mM EDTA, 1% Triton X-100, 0.1% SDS and 1% β-mercaptoethanol at37° C. for 24 hours. Samples were eluted by boiling for 5 minutes in theSDS-sample buffer, and analyzed by SDS-polyacrylamide gelelectrophoresis as described above. Hydrolysis of N-linked carbohydratesby endoglycosidase F shifted the 53 kDa band to 51 kDa. The extracelluardomain of ALK-5 contains one potential acceptor site for N-glycosylationand the size of the deglycosylated protein is close to the predictedsize of the core protein.

Establishment of PAE Cell Lines Expressing ALK-5

In order to investigate whether the ALK-5 cDNA encodes a receptor forTGF-β, porcine aortic endothelial (PAE) cells were transfected with anexpression vector containing the ALK-5 cDNA, and analyzed for thebinding of ¹²⁵I-TGF-β1.

PAE cells were cultured in Ham's F-12 medium supplemented with 10% FBSand antibiotics (Miyazono et al., (1988) J. Biol. Chem. 263, 6407-6415).The ALK-5 cDNA was cloned into the cytomegalovirus (CMV)-basedexpression vector pcDNA I/NEO (Invitrogen), and transfected into PAEcells by electroporation. After 48 hours, selection was initiated byadding Geneticin (G418 sulphate; Gibco—BRL) to the culture medium at afinal concentration of 0.5 mg/ml (Westermark et al, (1990) Proc. Natl.Acad. Sci. USA 87, 128-132). Several clones were obtained, and afteranalysis by immunoprecipitation using the VPN antiserum, one clonedenoted PAE/TβR-1 was chosen and further analyzed.

Iodination of TGF-β1, Binding and Affinity Crosslinking

Recombinant human TGF-β1 was iodinated using the chloramine T methodaccording to Frolik et al., (1984) J. Biol. Chem. 259, 10995-11000.Cross-linking experiments were performed as previously described (Ichijoet al., (1990) Exp. Cell Res. 187, 263-269). Briefly, cells in 6-wellplates were washed with binding buffer (phosphate-buffered salinecontaining 0.9 mM CaCl₂, 0.49 mM MgCl₂ and 1 mg/ml bovine serum albumin(BSA)), and incubated on ice in the same buffer with ¹²⁵I-TGF-β1 in thepresence or absence of excess unlabelled TGF-β1 for 3 hours. Cells werewashed and cross-linking was done in the binding buffer without BSAtogether with 0.28 mM disuccinimidyl suberate (DSS; Pierce Chemical Co.)for 15 minutes on ice. The cells were harvested by the addition of 1 mlof detachment buffer (10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 10% glycerol,0.3 mM PMSF). The cells were pelleted by centrifugation, thenresuspended in 50 μl of solubilization buffer (125 mM NaCl, 10 mMTris-HCl, pH 7.4, 1 mM EDTA, 1% Triton X-100, 0.3 mM PMSF, 1% Trasylol)and incubated for minutes on ice. Cells were centrifuged again andsupernatants were subjected to analysis by SDS-gel electrophoresis using4-15% polyacrylamide gels, followed by autoradiography. ¹²⁵I-TGF-β1formed a 70 kDa cross-linked complex in the transfected PAE cells(PAE/TβR-I cells). The size of this complex was very similar to that ofthe TGF-β type I receptor complex observed at lower amounts in theuntransfected cells. A concomitant increase of 94 kDa TGF-β type IIreceptor complex could also be observed in the PAE/TfiR-I cells.Components of 150-190 kDa, which may represent crosslinked complexesbetween the type I and type II receptors, were also observed in thePAE/TβR-I cells.

In order to determine whether the cross-linked 70 kDa complex containedthe protein encoded by the ALK-5 cDNA, the affinity cross-linking wasfollowed by immunoprecipitation using the VPN antiserum. For this, cellsin 25 cm² flasks were used. The supernatants obtained aftercross-linking were incubated with 7 μl of preimmune serum or VPNantiserum in the presence or absence of 10 μg of peptide for 1.5 h at 4°C. Immune complexes were then added to 50 μl of protein A-Sepharoseslurry and incubated for 45 minutes at 4° C. The protein A-Sepharosebeads were washed four times with the washing buffer, once withdistilled water, and the samples were analyzed by SDS-gelelectrophoresis using 4-15% polyacrylamide gradient gels andautoradiography. A 70 kDa cross-linked complex was precipitated by theVPN antiserum in PAE/TβR-1 cells, and a weaker band of the same size wasalso seen in the untransfected cells, indicating that the untransfectedPAE cells contained a low amount of endogenous ALK-5. The 70 kDa complexwas not observed when preimmune serum was used, or when immune serum wasblocked by 10 μg of peptide. Moreover, a coprecipitated 94 kDa componentcould also be observed in the PAE/TBR-I cells. The latter component islikely to represent a TGF-β type II receptor complex, since anantiserum, termed DRL, which was raised against a synthetic peptide fromthe C-terminal part of the TGF-β type II receptor, precipitated a 94 kDaTGF-β type II receptor complex, as well as a 70 kDa type I receptorcomplex from PAE/TβR-I cells.

The carbohydrate contents of ALK-5 and the TGF-β type II receptor werecharacterized by deglycosylation using endoglycosidase F as describedabove and analyzed by SDS-polyacrylamide gel electrophoresis andautoradiography. The ALK-5 cross-linked complex shifted from 70 kDa to66 kDa, whereas that of the type II receptor shifted from 94 kDa to 82kDa. The observed larger shift of the type II receptor band comparedwith that of the ALK-5 band is consistent with the deglycosylation dataof the type I and type II receptors on rat liver cells reportedpreviously (Cheifetz et al (1988) J. Biol. Chem. 263, 16984-16991), andfits well with the fact that the porcine TGF-β type II receptor has twoN-glycosylation sites (Lin et al (1992) Cell 68, 775-785), whereas ALK-5has only one (see SEQ ID No. 9).

Binding of TGF-β1 to the type I receptor is known to be abolished bytransient treatment of the cells with dithiothreitol (DTT) (Cheifetz andMassague (1991) J. Biol. Chem. 266, 20767-20772; Wrana et al (1992) Cell71, 1003-1014). When analyzed by affinity cross-linking, binding of¹²⁵I-TGF-β1 to ALK-5, but not to the type II receptor, was completelyabolished by DTT treatment of PAE/TβR-1 cells. Affinity cross-linkingfollowed by immunoprecipitation by the VPN antiserum showed that neitherthe ALK-5 nor the type II receptor complexes was precipitated after DTTtreatment, indicating that the VPN antiserum reacts only with ALK-5. Thedata show that the VPN antiserum recognizes a TGF-β type I receptor, andthat the type I and type II receptors form a heteromeric complex.

¹²⁵I-TGF-1 Binding & Affinity Crosslinking of Transfected COS Cells

Transient expression plasmids of ALKs-1 to -6 and TβR-II were generatedby subcloning into the pSV7d expression vector or into the pcDNA Iexpression vector (Invitrogen). Transient transfection of COS-1 cellsand iodination of TGF-β1 were carried out as described above.Crosslinking and immunoprecipitation were performed as described for PAEcells above.

Transfection of cDNAs for ALKs into COS-1 cells did not show anyappreciable binding of ¹²⁵I-TGFβ1, consistent with the observation thattype I receptors do not bind TGF-β in the absence of type II receptors.When the TβR-II cDNA was co-transfected with cDNAs for the differentALKs, type I receptor-like complexes were seen, at different levels, ineach case. COS-1 cells transfected with TβR-II and ALK cDNAs wereanalyzed by affinity crosslinking followed by immunoprecipitation usingthe DRL antisera or specific antisera against ALKs. Each one of the ALKsbound ¹²⁵I-TGF-β1 and was coimmunoprecipitated with the TβR-II complexusing the DRL antiserum. Comparison of the efficiency of the differentALKs to form heteromeric complexes with TβR-II, revealed that ALK-5formed such complexes more efficiently than the other ALKs. The size ofthe crosslinked complex was larger for ALK-3 than for other ALKs,consistent with its slightly larger size.

Expression of the ALK Protein in Different Cell Types

Two different approaches were used to elucidate which ALK's arephysiological type I receptors for TGF-β.

Firstly, several cell lines were tested for the expression of the ALKproteins by cross-linking followed by immunoprecipitation using thespecific antiseras against ALKs and the TGF-β type II receptor. The minklung epithelial cell line, MvlLu, is widely used to provide target cellsfor TGF-β action and is well characterized regarding TGF-β receptors(Laiho et al (1990) J. Biol. Chem. 265, 18518-18524; Laiho et al (1991)J. Biol. Chem. 266, 9108-9112). Only the VPN antiserum efficientlyprecipitated both type I and type II TGF-β receptors in the wild typeMvlLu cells. The DRL antiserum also precipitated components with thesame size as those precipitated by the VPN antiserum. A mutant cell line(R mutant) which lacks the TGF-β type I receptor and does not respond toTGF-β (Laiho et al, supra) was also investigated by cross-linkingfollowed by immunoprecipitation. Consistent with the results obtained byLaiho et al (1990), supra the type III and type II TGF-β receptorcomplexes, but not the type I receptor complex, were observed byaffinity crosslinking. Crosslinking followed by immunoprecipatitionusing the DRL antiserum revealed only the type II receptor complex,whereas neither the type I nor type II receptor complexes was seen usingthe VPN antiserum. When the cells were metabolically labelled andsubjected to immunoprecipitation using the VPN antiserum, the 53 kDaALK-5 protein was precipitated in both the wild-type and R mutant MvlLucells. These results suggest that the type I receptor expressed in the Rmutant is ALK-5, which has lost the affinity for binding to TGF-β aftermutation.

The type I and type II TGF-β receptor complexes could be precipitated bythe VPN and DRL antisera in other cell lines, including human foreskinfibroblasts (AG1518), human lung adenocarcinoma cells (A549), and humanoral squamous cell carcinoma cells (HSC-2). Affinity cross-linkingstudies revealed multiple TGF-β type I receptor-like complexes of 70-77kDa in these cells. These components were less efficiently competed byexcess unlabelled TGF-β1 in HSC-2 cells. Moreover, the type II receptorcomplex was low or not detectable in A549 and HSC-cells. Cross-linkingfollowed by immunoprecipitation revealed that the VPN antiserumprecipitated only the 70 kDa complex among the 70-77 kDa components. TheDRL antiserum precipitated the 94 kDa type II receptor complex as wellas the 70 kDa type I receptor complex in these cells, but not theputative type I receptor complexes of slightly larger sizes. Theseresults suggest that multiple type I TGF-β receptors may exist and thatthe 70 kDa complex containing ALK-5 forms a heteromeric complex with theTGF-β type II receptor cloned by Lin et al (1992) Cell 68, 775-785, moreefficiently that the other species. In rat pheochromocytoma cells (PC12)which have been reported to have no TGF-β receptor complexes by affinitycross-linking (Massague et al (1990) Ann. N.Y. Acad. Sci. 593, 59-72),neither VPN nor DRL antisera precipitated the TGF-β receptor complexes.The antisera against ALKs-1 to -4 and ALK6 did not efficientlyimmunoprecipitate the crosslinked receptor complexes in porcine aorticendothelial (PAE) cells or human foreskin fibroblasts.

Next, it was investigated whether ALKs could restore responsiveness toTGF-β in the R mutant of MvlLu cells, which lack the ligand-bindingability of the TGF-β type I receptor but have intact type II receptor.Wild-type MvlLu cells and mutant cells were transfected with ALK cDNAand were then assayed for the production of plasminogen activatorinhibitor-1 (PAI-1) which is produced as a result of TGF-β receptoractivation as described previously by Laiho et al (1991) Mol. Cell Biol.11, 972-978. Briefly, cells were added with or without 10 ng/ml ofTGF-β1 for 2 hours in serum-free MCDB 104 without methionine.Thereafter, cultures were labelled with [³⁵S] methionine (40 μCi/ml) for2 hours. The cells were removed by washing on ice once in PBS, twice in10 mM Tris-HCl (pH 8.0), 0.5% sodium deoxycholate, 1 mM PMSF, twice in 2mM Tris-HCl (pH 8.0), and once in PBS. Extracellular matrix proteinswere extracted by scraping cells into the SDS-sample buffer containingDTT, and analyzed by SDS-gel electrophoresis followed by fluorographyusing Amplify. PAI-1 can be identified as a characteristic 45kDa band(Laiho et al (1991) Mol. Cell Biol. 11, 972-978). Wild-type MvlLu cellsresponded to TGF-β and produced PAI-1, whereas the R mutant clone didnot, even after stimulation by TGF-β1. Transient transfection of theALK-5 cDNA into the R mutant clone led to the production of PAI-1 inresponse to the stimulation by TGF-β1, indicating that the ALK-5 cDNAencodes a functional TGF-β type I receptor. In contrast, the R mutantcells that were transfected with other ALKs did not produce PAI-1 uponthe addition of TGF-β1.

Using similar approaches as those described above for the identificationof TGF-β-binding ALKs, the ability of ALKs to bind activin in thepresence of ActRII was examined. COS-1 cells were co-transfected asdescribed above. Recombinant human activin A was iodinated using thechloramine T method (Mathews and Vale (1991) Cell 65, 973-982).Transfected COS-1 cells were analysed for binding and crosslinking ofI-activin A in the presence or absence of excess unlabelled activin A.The crosslinked complexes were subjected to immunoprecipitation usingDRL antisera or specific ALK antisera.

All ALKs appear tc bind activin A in the presence of Act R-II. This ismore clearly demonstrated by affinity cross-linking followed byimmunopreciptation. ALK-2 and ALK-4 bound ¹²⁵I-activin A and werecoimmunoprecipitated with ActR-II. Other ALKs also bound 125I-activin Abut with a lower efficiency compared to ALK-2 and ALK-4.

In order to investigate whether ALKs are physiological activin type Ireceptors, activin responsive cells were examined for the expression ofendogenous activin type I receptors. MvlLu cells, as well as the Rmutant, express both type I and type II receptors for activin, and the Rmutant cells produce PAI-1 upon the addition of activin A. MvlLu cellswere labeled with ¹²⁵I-activin A, cross-linked and immunoprecipitated bythe antisera against ActR-II or ALKs as described above.

The type I and type II receptor complexes in MvlLu cells wereimmunoprecipitated only by the antisera against ALK-2, ALK-4 andActR-II. Similar results were obtained using the R mutant cells. PAEcells do not bind activin because of the lack of type II receptors foractivin, and so cells were transfected with a chimeric receptor, toenable them to bind activin, as described herein. A plasmid (chim A)containing the extracelluar domain and C-terminal tail of Act R-II(amino-acids −19 to 116 and 465 to 494, respectively (Mathews and Vale(1991) Cell, 65., 973-982)) and the kinase domain of TBR-II (amino-acids160-543) (Lin et al (1992) Cell, 68, 775-785) was constructed andtransfected into pcDNA/neo (Invitrogen). PAE cells were stablytransfected with the chim A plasmid by electroporation, and cellsexpressing the chim A protein were established as described previously.PAE/Chim A cells were then subjected to ¹²⁵I-activin A labellingcrosslinking and immunoprecipitation as described above.

Similar to MvlLu cells, activin type I receptor complexes in PAE/Chim Acells were immunoprecipitated by the ALK-2 and ALK-4 antisera. Theseresults show that both ALK-2 and ALK-4 serve as high affinity type Ireceptors for activin A in these cells.

ALK-1, ALK-3 and ALK-6 bind TGF-β1 and activin A in the presence oftheir respective type II receptors, but the functional consequences ofthe binding of the ligands remains to be elucidated.

The invention has been described by way of example only, withoutrestriction of its scope. The invention is defined by the subject matterherein, including the claims that follow the immediately following fullSequence Listings.

1. An isolated antibody which binds specifically to an amino acidsequence encoded by the nucleotide sequence set forth in SEQ ID NO: 5(ALK-3).
 2. The isolated antibody of claim 1, wherein the antibody bindspecifically to the amino sequence of SEQ ID NO:
 6. 3. The isolatedantibody of claim 1, wherein the antibody binds specifically to anextracellular domain of a human protein encoded by the nucleotidesequence set forth in SEQ ID NO:
 5. 4. The isolated antibody of claim 1,wherein the antibody binds specifically to amino acids 181-202 of SEQ IDNO: 6.